Natural killer (NK) cells comprise a subset of lymphocytes involved in protection against microbial pathogens and tumors. Because of their cytotoxic capacity, NK cells are able to directly eliminate abnormal cells. Lytic granules of NK cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Defects in lytic granule secretion are associated with serious diseases, such as hemophagocytic lymphohistiocytosis and Chediak-Higashi, Hermansky-Pudlak, or Griscelli syndromes. Knowledge about the proteins involved in regulation of lytic granule release is very limited, and the granule-specific protein machinery involved in NK-cell exocytosis is poorly understood. Lytic granules have the characteristics of lysosomes and are referred to as secretory lysosomes. There are many proteins residing in lysosomal membranes that function in acidification of the lysosomal lumen, protein import, membrane fusion, and transport of degradation products to the cytoplasm. Surprisingly, the role of the most abundant proteins, lysosome-associated membrane protein (LAMP) 1 and LAMP2 (CD107a and CD107b, respectively), is ill-defined. In humans, mutations in LAMP2 cause Danon disease, believed to arise from aberrant autophagy. LAMP2-deficient mice also accumulate autophagic vacuoles in several tissues. LAMP1-deficient mice have an almost normal phenotype but have elevated levels of LAMP2, suggesting that LAMP2 can compensate for the loss of LAMP1. Recent studies using LAMP-depleted fibroblasts demonstrate that both proteins are required for proper lysosomal transport and efficient (auto)phagosome-lysosome fusion. LAMP1 is also widely used as a marker for the identification of NK-cell degranulation, as it appears on the cell surface following the fusion of lysosomes with the plasma membrane, but the role of LAMP1 in NK-cell biology is completely unknown. Since LAMP1 is one of the most abundant proteins in the lysosomal membrane and NK cells use their lysosomes to induce the death of target cells, we sought to determine the importance of LAMP1 in NK-cell cytolytic activity. Therefore, this project emphasis has been to understand the role of lysosomal membrane protein 1, LAMP1, in regulation of human NK cell cytotoxicity. To address this issue, RNA interference (RNAi) was used to disrupt LAMP1 expression in human NK cell lines, as well as primary NK cells, and the effects of LAMP1 silencing on lytic granule composition, movement, exocytosis and NK cell cytotoxic potential were investigated. We have demonstrated that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNAi cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi networkderived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity. Thus, we have shown that disruption of LAMP1 expression has a pleiotropic effect on NK cells, causing impairment of traf&#64257;cking of a critical lytic granule protein, perforin, and slower movement of lytic granules. This results in the inability of NK cells to deliver granzyme B to target cells and inhibition of NK-cell cytotoxicity. Thus, LAMP1 is not only a marker of NK-cell degranulation, but also a crucial component of NK-cell cytotoxicity.